All cells must rapidly and accurately replicate their DNA during the process of cell division. DNA replication is carried out by complex multi-protein assemblies, which must be recruited to and loaded onto the template DNA in a highly coordinated fashion. The DNA polymerase III (Pol III) holoenzyme from the bacterium E. coli is a model system for studies of DNA replication. A key component of the Pol III holoenzyme is the 5 subunit DnaX complex, which has known functions in loading the "sliding clamp" processivity factor onto the DNA and in coordinating leading and lagging strand synthesis. One of the DnaX subunits, the product of the DnaX gene, occurs in vivo as either a full length or truncated version. While both forms support processive DNA synthesis, there are important functional differences between the two components. The goal of this proposal is to ascertain how the two forms of the DnaX protein affect the mechanism for initiating DNA replication and to thereby better understand the role of the DnaX complex. Rapid kinetic assays will be developed to measure the rate for assembling the Pol III holoenzyme on a DNA template. Both a KinTek rapid mix/quench device and fluorescence resonance energy transfer techniques will be utilized. The kinetic advantage of the full length DnaX protein over the truncated version will be established experimentally. The mechanism by which holoenzyme/DNA initiation complexes are formed will be elaborated by examining the kinetic effects as the concentration of each holoenzyme component is varied. These experiments will test the hypothesis that the core Pol III DNA polymerase is "chaperoned" to the initiation complex by DnaX complexes containing the full length DnaX protein. PUBLIC HEALTH RELEVANCE The fast and accurate replication of genetic information is a fundamental process of cell division. Thus, research that deepens our understanding of this process is crucial for developing more effective treatments for cancer or pathogenic infection. The results of this proposal will establish the role of a key component in the DNA replication machinery and will lead to a greater understanding of how DNA replication is initiated at the molecular level.